Journal: The Journal of Biological Chemistry

Article Title: The pH-dependent Client Release from the Collagen-specific Chaperone HSP47 Is Triggered by a Tandem Histidine Pair *

doi: 10.1074/jbc.M115.706069

Figure Lengend Snippet: The interaction of wild-type HSP47 and various histidine mutants with collagen as measured by biolayer interferometry. A foldon-stabilized collagen mimetic peptide was loaded on a streptavidin chip and incubated at constant concentration (5 μm) at the indicated pH values. Association was measured for 60 s, and dissociation time varied between 80 and 140 s (only the first 80 s are shown). koff values were fitted by a 1:1 Langmuir model. All dissociation curves show a clear dependence of the koff with the pH. Most mutants behave similarly to the wild-type protein. However, H215N and H215N/H216N have an overall faster dissociation rate, whereas the dissociation rates of H273N/H274N, H273A/H274A, and to some degree H273A change less with pH. Data show representative curves from at least three technical replicates. Results for wild type, H273N, H274N, and H273N/H274N were confirmed using at least three independent biological samples.

Article Snippet: For coupling on streptavidin biosensors, foldon peptides were biotinylated with NHS-Biotin following the manufacturer's protocols (Pierce) and loaded at a concentration of 10 μ m to a response of ∼5.5 nm.

Techniques: Incubation, Concentration Assay

Journal: The Journal of Biological Chemistry

Article Title: The pH-dependent Client Release from the Collagen-specific Chaperone HSP47 Is Triggered by a Tandem Histidine Pair *

doi: 10.1074/jbc.M115.706069

Figure Lengend Snippet: Kinetic analysis of the biolayer interferometry data. A foldon-stabilized collagen mimetic peptide was loaded on a streptavidin chip and incubated at constant concentration (5 μm) with HSP47 variants at the indicated pH values. Kinetic parameters were extracted using a 1:1 Langmuir binding model. For wild type and most variants, there is a clear increase in koff with decreasing pH observable, which is typically about 5-fold, as seen in A (top left). The histidine pair previously assigned as being responsible for the pH switch, His215 and His216, shows minor changes in pH dependence upon double mutation to asparagine (A, top right). However, the binding of the H215N/H216N double mutant is weaker than the wild type. The H273N/H274N double mutant has a very much decreased dependence of koff (A, bottom left) and KD (C) on the pH value, and the H273A/H274A variant behaves very similarly (A, bottom right). The kon values are less sensitive to pH changes (B). Error bars, S.D.

Article Snippet: For coupling on streptavidin biosensors, foldon peptides were biotinylated with NHS-Biotin following the manufacturer's protocols (Pierce) and loaded at a concentration of 10 μ m to a response of ∼5.5 nm.

Techniques: Incubation, Concentration Assay, Binding Assay, Mutagenesis, Variant Assay

Journal: The Journal of Biological Chemistry

Article Title: The pH-dependent Client Release from the Collagen-specific Chaperone HSP47 Is Triggered by a Tandem Histidine Pair *

doi: 10.1074/jbc.M115.706069

Figure Lengend Snippet: Analysis of the interaction of HSP47 H238N with collagen. A, interaction of HSP47 WT (blue, cyan, and green sensorgrams) and H238N (yellow, orange, and red) was measured in biolayer interferometry. A foldon-stabilized collagen mimetic peptide was loaded on a streptavidin chip and incubated at the indicated concentrations. For H238N, no significant signal even at the highest concentration could be detected. B, in the crystal structure, His238 can interact via a hydrogen bond with Asp220, which stabilizes Arg222 (blue dashed lines). Arg222 and Asp385 make important contacts to the collagen helix (green and yellow dashed lines). C, representative snapshot of the uncomplexed H238N mutant obtained from an constant pH MD simulation, revealing a rearrangement of the hydrogen bonding network compared with the wild type; Asn238 now interacts with Ser305 (red dashed line), and the Asp220-Arg222 salt bridge is lost. Arg222 forms a stable salt bridge with Asp385 (green dashed lines), both important residues for collagen recognition. HSP47 is shown in ribbon (cyan/gray) and collagen as sticks in orange, and all numbers are according to full-length canine protein. R#8, arginine in the collagen model peptide crystallized (Protein Data Bank entry 4AU2). D–F, comparison of the conformational stability of several side-chain interactions in MD simulations between wild type and H238N HSP47. For wild-type HSP47, two plots are shown in each panel corresponding to the unliganded and collagen-bound form. Black and gray, wild-type protein with and without collagen, respectively. Blue, red, and green triangles, distances between the indicated atoms in the H238N mutant. Biolayer interferometry data show representative curves from three technical replicates. The general result was confirmed in three independent biological replicates.

Article Snippet: For coupling on streptavidin biosensors, foldon peptides were biotinylated with NHS-Biotin following the manufacturer's protocols (Pierce) and loaded at a concentration of 10 μ m to a response of ∼5.5 nm.

Techniques: Incubation, Concentration Assay, Mutagenesis

Journal: The Journal of Biological Chemistry

Article Title: The pH-dependent Client Release from the Collagen-specific Chaperone HSP47 Is Triggered by a Tandem Histidine Pair *

doi: 10.1074/jbc.M115.706069

Figure Lengend Snippet: The influence of charged residues at positions 273 and 274 on collagen binding. A foldon-stabilized collagen mimetic peptide was loaded on a streptavidin chip and incubated at constant concentration (5 μm) with HSP47 variants at the indicated pH values (A). Kinetic parameters were extracted using a 1:1 Langmuir binding model. KD for H274K at pH 6.0 is not visible at the used scale (B–D). Introduction of the negatively charged Asp at either position, 273 or 274, reduces the pH dependence of koff. Interestingly, also the H273K mutant shows a slightly tighter binding to collagen at low pH. Data show representative curves from at least three technical replicates. Results were confirmed using at least two independent biological samples.

Article Snippet: For coupling on streptavidin biosensors, foldon peptides were biotinylated with NHS-Biotin following the manufacturer's protocols (Pierce) and loaded at a concentration of 10 μ m to a response of ∼5.5 nm.

Techniques: Binding Assay, Incubation, Concentration Assay, Mutagenesis

Journal: The Journal of Biological Chemistry

Article Title: The pH-dependent Client Release from the Collagen-specific Chaperone HSP47 Is Triggered by a Tandem Histidine Pair *

doi: 10.1074/jbc.M115.706069

Figure Lengend Snippet: The effect of single amino acid exchanges present in various fish species on collagen binding. A foldon-stabilized collagen mimetic peptide was loaded on a streptavidin chip and incubated at constant concentration (5 μm) with HSP47 variants at the indicated pH values. Kinetic parameters were extracted using a 1:1 Langmuir binding model. The pH dependence of HSP47 WT and H273Y are indistinguishable. H273L and H273F, however, have an intermediate pH dependence similar to that of H273A (Fig. 6). Data show representative fits from at least three technical replicates. Results were confirmed using at least two independent biological samples. A, dissociation rate constant (kOFF). B, association rate constant (kON). C, dissociation constant (KD) calculated as kOFF/kON.

Article Snippet: For coupling on streptavidin biosensors, foldon peptides were biotinylated with NHS-Biotin following the manufacturer's protocols (Pierce) and loaded at a concentration of 10 μ m to a response of ∼5.5 nm.

Techniques: Binding Assay, Incubation, Concentration Assay